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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 5  |  Issue : 1  |  Page : 35-40

Remineralization potential of varnish containing casein phosphopeptides-amorphous calcium phosphate with fluoride and varnish containing only fluoride: A comparative study


1 Department of Dentistry, Hassan Institute of Medical Sciences, Hassan, India
2 Department of Pedodontics and Preventive Dentistry, The Oxford Dental College, Hospital and Research Centre, Bengaluru, Karnataka, India

Date of Web Publication12-Mar-2018

Correspondence Address:
Priya Subramaniam
Department of Pedodontics and Preventive Dentistry, The Oxford Dental College and Hospital, Bengaluru - 560 068, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/sjos.SJOralSci_44_17

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  Abstract 


Background: Fluoride varnishes have been the standard of practice for the professional application of fluoride. The primary reason for wide acceptance of fluoride varnish is that the procedure is easy, safe, convenient, and well-accepted by patients.
Aim: The aim of this study is to evaluate and compare the remineralizing potential of varnish containing casein phosphopeptides-amorphous calcium phosphate (CPP-ACP) and fluoride (MI Varnish®), with that of varnish containing only fluoride (Fluor Protector®).
Materials and Methods: Fifty premolar teeth samples were used for this part of the study. They were divided into two Groups (1 and 2), consisting of 25 samples each. Two windows on the buccal surface of these samples were made and assigned randomly as control and experimental window. Initial depth of carious lesions of both windows was measured in all the samples. No varnish was applied on control windows. On the experimental window of Subgroup 1, varnish containing CPP-ACP with fluoride was applied, and on samples of Group 2, varnish containing only fluoride was applied. After varnish application, all the samples were subjected to pH-cycling. Following, the final depth of carious lesion was calculated using polarized light microscope. The data obtained were tabulated and subjected to statistical analysis.
Results: The final depths of artificial carious lesion of experimental window of Group 1 and 2 were 131.26 ± 7.01 μm and 134.11 ± 5.95 μm, respectively.
Conclusion: There was no significant difference in the remineralizing potential of varnish containing CPP-ACP and fluoride and varnish containing only fluoride.

Keywords: Casein phosphopeptides-amorphous calcium phosphate, fluoride, remineralization potential, varnish


How to cite this article:
Girish Babu K L, Subramaniam P, Teleti S. Remineralization potential of varnish containing casein phosphopeptides-amorphous calcium phosphate with fluoride and varnish containing only fluoride: A comparative study. Saudi J Oral Sci 2018;5:35-40

How to cite this URL:
Girish Babu K L, Subramaniam P, Teleti S. Remineralization potential of varnish containing casein phosphopeptides-amorphous calcium phosphate with fluoride and varnish containing only fluoride: A comparative study. Saudi J Oral Sci [serial online] 2018 [cited 2018 Aug 15];5:35-40. Available from: http://www.saudijos.org/text.asp?2018/5/1/35/227131




  Introduction Top


Fluoride varnishes have been the standard of practice for the professional application of fluoride. The primary reason for wide acceptance of fluoride varnish is that the procedure is easy, safe, convenient, and well accepted by patients. Patient's exposure to fluoride can be better controlled with varnishes, and less chair time is required than that needed for conventional foams and gels.[1] Fluoride varnish covers the teeth with an adherent film that lasts for up to 24 h, which is the recommended time before a patient can resume brushing, thereby enhancing the uptake of fluoride ions into the tooth structure. Fluoride is deposited as calcium fluoride, creating a reservoir of fluoride ions, which are slowly released. Thus, the action of fluoride is related to its inhibition of the demineralization as well as its promotion of enamel remineralization.[2]

Duraphat® varnish alone achieves significant remineralization of enamel carious lesions.[3] Xylitol varnishes seem to be promising alternatives to increase enamel remineralization in situ.[4] Although fluoride has a profound anticaries effect, high fluoride strategy cannot be followed in most instances to avoid the potential for adverse effects due to overexposure to fluoride.[5] This need has redirected research to develop novel preventive agents that can act as an adjunct to fluoride or independent of it. Casein phosphopeptides-amorphous calcium phosphate (CPP-ACP) is one such agent that has been proposed to have anticariogenic properties. CPP is a sticky protein that binds and stabilizes calcium and phosphate ions in an amorphous state.[6] CPP-ACP has been shown to localize to tooth surfaces and during acidogenic challenges may release calcium, phosphate and fluoride, and maintain the supersaturation of calcium and phosphate ions in the vicinity of the tooth surface, and thereby depressing demineralization and enhancing remineralization.[7]

Fluoride varnishes containing ACP, tricalcium phosphate, and sodium calcium phosphosilicate are all promising methods to inhibit demineralization and to increase remineralization. The treatment of dental caries may be improved by the application of these enhanced varnishes. Varnish containing CPP-ACP with fluoride improves the surface microhardness of enamel. Furthermore, prolonged use of these varnishes decreases the caries incidence in children.[8] Studies have shown that remineralization potential of CPP-ACP combined with fluoride has been more effective.[9],[10],[11],[12]

Commercially, CPP-ACP has been incorporated into chewing gums, dentifrices, pastes, crèmes, lozenges, and rinses. Studies that reported the benefits of the synergistic effect of CPP-ACP with fluoride were carried out on dentifrices.[7],[13],[14] Recently, a combination of CPP-ACP with fluoride has been introduced as a dental varnish-MI Varnish®. Hence, the aim of the present study was to evaluate and compare the remineralizing potential of this newly introduced varnish containing CPP-ACP and fluoride (MI Varnish®), with that of varnish containing only fluoride (Fluor Protector®).


  Materials and Methods Top


Ethical clearance to conduct the study was obtained from the institutional review board before commencement of the study. Premolar teeth samples extracted for the purpose of orthodontic treatment were collected from normal, healthy children aged 13–15 years. Written informed consent was obtained from patients as well as parents for the use of these teeth in this particular study. Inclusion criteria: 1. Teeth with intact enamel surfaces, 2. Teeth not affected by fluorosis, 3. Teeth without white spot lesions, and 4. Teeth with no signs of decalcification. Exclusion criteria: 1. Teeth with caries, 2. Teeth with developmental defects, and 3. Teeth with signs of fracture. A total of 63 teeth were collected. The soft-tissue deposits and calculus were removed from the teeth with a surface scaler. Teeth were cleaned using a slurry of pumice. All teeth were then stored in distilled water containing 0.2% thymol to inhibit the microbial growth until the study was carried out.

The collected 63 teeth samples were coated with acid-resistant nail varnish (Revlon®, New York, USA), leaving two square windows of 2 mm2 × 2 mm2 each on the buccal surface. The advantage of this design was the depth of the lesion can be determined in any tooth at baseline, minimizing variations in initial lesion depth among specimens. Each tooth was immersed in 3-ml demineralizing solution for 4 days to create artificial enamel lesions. On the 5th day, all the samples were taken out of the demineralization solution and were dried using blotting paper. The initial depth of the artificial carious lesion was measured using polarized light microscope (Nikon® model eclipse E400 pol, Tokyo, Japan) at ×10 magnification. Teeth with lesion depth ranging between 100 and 150 μm were included.[15] Among 63 samples, only 50 samples fulfilled the criteria of desired lesional depth, and 13 samples were excluded from the study.

These 50 teeth samples were divided into two groups, namely, Group 1 and 2, consisting of 25 samples each. The two windows on the buccal surface were assigned randomly by the toss of a coin as control and experimental window. The initial depth of carious lesions of both windows was measured at three points using polarized light microscope at ×10 magnification.[16]

No varnish was applied on control windows. On the experimental window of teeth samples of Group 1, varnish containing CPP-ACP with fluoride (MI Varnish®, GC India Dental Pvt Ltd., India) was applied, and on teeth samples of Group 2, varnish containing only fluoride (Fluor Protector®, Ivoclar Vivadent, Amherst, N. J, USA) was applied. Following, all the teeth samples were subjected to 5-day pH-cycling.[17] On the 6th day, all the samples were taken out of the remineralization solution and dried using blotting paper, and the final depth of carious lesion was calculated using polarized light microscope at ×10 magnification.[17]

Photographs of the final carious lesions were taken for both the windows to calculate the depth of the carious lesion. Photographs were analyzed using a computerized calculation method with Image-Pro® Plus (Media Cybernetics, Silver Spring, MD). The maximum depth of the lesion was measured at three points (D1, D2, and D3) to avoid errors and the results were averaged.[16] The values were expressed in micrometers (μm). The data obtained were tabulated and subjected to statistical analysis using Student's t-test. P value was fixed at P ≤ 0.05 for significance testing and P < 0.001 was considered as highly significant.


  Results Top


The control window of Group 1 teeth samples showed the initial and final depth of artificial carious lesion of 131.07 ± 7.25 and 131.07 ± 7.25, respectively. This difference was statistically significant (P = 0.0053). In Group 2 teeth samples, it was 131.75 ± 6.75 (initial depth) and 146.95 ± 7.13 (final depth) and the difference was statistically significant (P = 0.0045). However, experimental windows in both groups did not show statistically significant difference [Table 1]. On comparison of the mean difference between control and experimental windows in Group 1, there was statistically significant difference (P = 0.0042). Even in Group 2, the mean difference was statistically significant (P = 0.0034) [Table 2]. The mean of final depth of artificial carious lesion in Group 1 was 131.26 ± 7.01, and in Group 2, it was 134.11 ± 5.95. The intercomparison of the mean difference between Group 1 and 2 was not statistically significant (P = 0.2) [Table 3].
Table 1: Comparison of initial and final depth of the artificial carious lesion between control and experimental windows in Group 1 and 2

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Table 2: Comparison of mean difference in initial and final depth of artificial carious lesion between control and experimental windows in Group 1 and 2

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Table 3: Comparison between final depths of artificial carious lesion of experimental window of Group 1 and 2

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  Discussion Top


Fluoride varnishes have been the standard of practice for the professional application of fluoride. Fluoride ions are deposited as calcium fluoride, creating a reservoir of fluoride ions, which are slowly released. Thus, the action of fluoride is related to its inhibition of the demineralization as well as its promotion of enamel remineralization.[2] The fluoride ions released by fluoridated varnish has a greater interaction with enamel and provide less mineral loss, thus, reducing enamel surface demineralization and carious lesion depth.[18] Fluoridated varnishes are effective in reducing or arresting the white spot lesion.[19] Other studies have also shown fluoride varnish to be clinically effective.[20],[21],[22]

The fluoride retention on enamel and subsurface lesion remineralization depends on the availability of the calcium and phosphate ions. However, combining calcium phosphate and fluoride ions can lead to loss of bioavailable fluoride ion due to a reaction between the calcium phosphate phase and the fluoride ion. In an approach to overcome this incompatibility of calcium phosphates and fluoride ions, various calcium phosphate technologies have been developed. One such approach is incorporating stable CPP and unstable ACP into dental products. The presence of the CPP stabilizes the ACP phase to deliver bioavailable calcium, phosphate, and fluoride ions to the tooth surface and promotes remineralization of carious lesions.[23] CPP-ACP paste decreases lesion depth and has higher remineralization potential when used in combination with fluoride toothpaste than when used alone.[24] Recently, the combination of CPP-ACP with fluoride has been introduced into a dental varnish (MI Varnish®). Very few studies have evaluated the efficacy of this newly introduced varnish.[25] Hence, the present study was taken up to evaluate remineralization potential of varnish containing CPP-ACP and fluoride (MI Varnish®) and compare with that of varnish containing only fluoride (Fluor Protector®).

The pH-cycling protocol followed in the present study was as described by Featherstone et al. The model is of particular interest because it simulates in vivo high caries risk condition and simultaneously measures the net result of the inhibition of demineralization and the enhancement of remineralization. In this model, the dynamic cycles of de- and re-mineralization are simulated by sequentially immersing enamel specimens in acidic (demineralizing) and supersaturated (remineralizing) buffer solutions. These de- and re-mineralization solutions approximate the mineral ion composition and supersaturation of saliva as originally reported by ten Cate and Duijsters.[15],[26] Polarized light microscopic analysis is an extremely sensitive technique to assess the remineralization potential of dental varnishes changes. With respect to de- and re-mineralization, it can qualitatively show the areas of mineral loss and mineral gain represented by the visualization of areas with different porosities and birefringence.

Remineralization of the artificial carious lesions may occur as a result of the deposition of the mineral into the porous zone rather than the emergence of the eroded crystals. The hydroxyapatite crystals are formed from the supersaturated calcium and phosphate ions, and the presence of fluoride ions is likely to further promote the remineralization process by forming fluorapatite crystals that are more resistant to future demineralization than hydroxyapatite.[27] In the present study, both the varnishes were able to remineralize the carious lesion effectively. Varnish containing CPP-ACP with fluoride showed higher remineralization potential than fluoridated varnish. Kumar et al. also observed higher remineralizing potential of CPP-ACP containing paste when applied as a topical coating after the use of a fluoridated toothpaste.[24] In an another study, CPP-ACP significantly provided better remineralizing effect than APF.[5] Majithia et al. observed that varnish with CPP-ACP with fluoride (MI Varnish™) to be better in remineralizing potential as compared to the other two tested varnishes.[28] Fluoride varnish containing tricalcium phosphate has also shown better remineralization capability than varnish without tricalcium phosphate.[29] Similarly, Clinpro™ White Varnish with Tri-Calcium phosphate was also effective in reducing demineralization in subsurface layer and in improving the remineralization of surface and subsurface enamel layer.[30] Llena et al. observed that daily topical application of CPP-amorphous calcium fluoride phosphate (ACFP) (MI Paste Plus®) to be superior in remineralizing the enamel smooth surface than monthly professional application of Duraphat® fluoride varnish.[31]

CPP-ACP stabilizes calcium, phosphate, and fluoride ions in a metastable form thus, facilitating high concentration of the calcium and phosphate ions which diffuses in the enamel lesion. Thus, CPP-ACP leads to increased incorporation of ions into subsurface enamel lesion and substantially increases remineralization of subsurface lesions compared to fluoride alone.[32] Due to this mechanism, there would have been increased remineralization in samples of Group 1. This highlights the importance of the availability of calcium and phosphate ions for net remineralization to occur. Cochrane et al. observed significant release of calcium and inorganic phosphate ions which is consistent with the bioavailable nature of CPP-ACP contained within the varnish. The fast release of the ions from this varnish under the relatively neutral conditions was attributed to the high water solubility of the CPP-ACP complexes.[25] Savas et al. also observed that CPP-ACP containing fluoride varnish (MI Varnish) provides remineralization of incipient carious lesions after a single application and suggested that it may be suitable for clinical use.[33] However, on comparison of remineralization potential of MI Varnish™ with that of Duraphat®, Embrace™ Varnish, Enamel Pro® Varnish, and Clinpro™ White Varnish, Duraphat® showed a superior remineralization potential than other tested varnishes.[3]

Fluoride ions reduce the enamel demineralization by forming new and larger fluorhydroxyapatite crystals and enhancing remineralization. At the enamel surface, the fluoride ions come in contact with free calcium and phosphate ions of de-remineralizing solutions, and fluorapatite is rapidly formed. Rapid deposition of fluorapatite crystals forms a firm surface layer, which is more resistant to further demineralization. However, at the same time, this surface layer is resistant to penetration of calcium and phosphate ions required to rebuild the lesion in depth.[34] Thus, remineralization by fluoride is a self-limiting surface phenomenon that permits the penetration of ions into the lesion only up to 80–110 μm. This could be the reason for the lower level of remineralization in samples of Group 2. The fluoride varnish added with silver nanoparticles seems to be more effective in the dental remineralization.[35] Application of fluoride varnish after irradiating the enamel surface with laser also enhances the remineralization.[36]

Although varnish containing CPP-ACP with fluoride showed a higher remineralization than fluoride varnish, it was not statistically significant. This may be due to the physical properties of the varnish used. The fluoride varnish applied on the tooth surface, evaporated quickly to form a thin film. Whereas, the varnish containing CPP-ACP and fluoride was more viscous because of which it may not have adequately wet the surface.[34] These reasons also explain the slight increase in the depth of the lesion observed in Group 1. An Indian study also observed a nonsignificant difference of remineralization potential between paste containing CPP-ACP with fluoride and fluoridated toothpaste.[16] However, Somasundaram et al. observed higher remineralization of enamel lesions with paste containing CPP-ACP than fluoridated toothpaste.[5] Oral hygiene instruction along with the application of fluoride varnish and CPP-ACP has also shown to be an effective method to reduce the demineralization. Prolonged use of CPP-ACP reduces dental caries in primary dentition.[8]

The results of the present study showed varnish containing CPP-ACP and fluoride is as effective as fluoride varnish. Although fluoride has a profound effect on the level of caries progression, high-fluoride strategy cannot be followed due to its adverse effects.[3] Thus, there is a need for nonfluoridated remineralizing agents or remineralizing agents with less fluoride. Hence, varnish containing CPP-ACP and fluoride can be effectively used as an alternative for fluoride varnish.

As this study was carried out under in vitro conditions on premolar teeth, the results may not be transferred completely to an in vivo situation. Therefore,in vivo studies on primary and permanent teeth have to be carried out to further evaluate the effectiveness of varnish containing CPP-ACP with fluoride.


  Conclusion Top


There was no significant difference in the remineralizing potential of varnish containing CPP-ACP and fluoride, with that of varnish containing only fluoride.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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